1. Technical Field
The present invention relates to the field of preservation of tissue for transplantation and more specifically to storage media that can extend tissue preservation.
2. Description of Related Art
Corneal transplantation is the most common form of organ transplantation practiced in the United States. Two procedures for transplantation are utilized. Penetrating keratoplasty is used in about 90% of the cases, with lamellar keratoplasty being utilized in the remaining situations. Demand for corneal transplantation exceeds the available supply of corneas. Advances in procurement and preservation must continue to meet this demand. JAMA 1988 Feb. 5; 259(5):719-22.
The need for corneal transplantation in the United States stems from conditions which effect the midstroma and endothelium. On a global basis the major blinding diseases stem from trachoma and vitamin A deficiency, both conditions of the superior stroma. Very often the areas in greatest need are places where steroids to modulate the immune response to the corneal transplant are unavailable and patient follow up is suboptimal for successful use for the lamellar grafting technique. Lamellar keratoplasty allows the corneal surgeon to transplant stroma and viable epithelial tissue while retaining the host endothelium and inferior stroma. The successful outcome of the surgery requires cornea with intact epithelium. Similarly, the Lasik Method requires cornea with intact, viable epithelium.
Perfluorocarbons
Perfluorocarbons are inert materials that were initially manufactured as part of the Manhattan project (Reiss) at the end of World War II. They were initially used to encase uranium. Perfluorocarbons are polymers of carbon, fluorine and hydrogen. They uniquely have cavities in which gaseous oxygen and carbon dioxide fit (Reiss, 1991). In the emulsion form, they have a larger surface area for gaseous exchange (Reiss, 1978; Dellacherie 1987; Parry 1988). Reviewed by Faithfull and Weers 1998 Vox Sang 74: Suppl 2:243-8.
Gollan and Clark reported in 1966 and 1967 that mice could survive for prolonged periods immersed in oxygenated perfluorocarbons. However, toxicity later attributed to impurities, preventing development of medical applications. In the early 1980's high quality pure perfluorocarbons and emulsions were developed. Perfluorocarbons are currently used in cardiac applications (Porter et al. Am. J. Cardiol. 1998 November 15:82(10) 1173-7. Liquid ventilation, i.e. postnatal long (Nobubara et al., 1998 J Pediatr Surg July 33(7) 1024-9, and artificial blood (Patel SASAIO) 1998 May-June: 44(3) 144-56; Cardiac Surgery Holman, et al., 1994 Artif Cells Blood Substit Immobil Biotechnol 22(4):979-90) and artificial blood uses of perfluorocarbons were pioneered by David Long in the late 1980's.
Perfluorocarbon liquids have been used in ophthalmology to facilitate surgery in a wide variety of conditions, including proliferative vitreoretinopathy, giant retinal tears, drainage of suprachoroidal hemorrhages, diabetic traction, retinal detachments with a rhegmatogenous component, dislocated crystalline or intraocular lenses, and retinal detachment associated with choroidal coloboma. The clarity of perfluorocarbon liquids, with a refractive index close to that of water, allows the use of a conventional contact lens for vitreous surgery while the low viscosity facilitates tissue manipulation, injection, and removal. All perfluorocarbon liquids when used as tamponading agents can compress and disorganize the retina. This “toxicity” is a physical effect rather than chemical toxicity and depends upon the amount of perfluorocarbon liquid injected. Perfluorocarbon liquids are not tolerated in the anterior chamber, causing corneal edema within two to three days at the site of contact. Peyman G Aet al. Surv. Ophthalmol 1995 March-April:39(5):375-95.
The concept of corneal transplantation is a hundred years old, the first successful transplant having been performed in 1897. The concept of increasing donor storage time by removing cadaver cornea and storing them in solutions was introduced by Filatov in 1937. Rycroft (Rycroft, 1954) and Beran (Beran, et al., 1958) extended corneal storage by placing cornea in liquid paraffin in the 1950's. Klen (Klen, et al., 1965) in the 1960's extended corneal transparency by insufflating air into the anterior chamber. In the early 1970's, cornea were stored in moist chambers for 24 hours.
The cornea is composed of five layers: epithelium, Bowmans layer, stroma, Descemets membrane and endothelium. It is a sandwich with the epithelium and endothelium being the bread slices and the stroma being the filling.
The stroma is made of parallel collagen fibrils arranged in lamellae (Edelhauser, et al., 1982; Tripathi et al., 1984; Maurice, 1984). These lamellar layers are arranged to create a pattern of destructive interference from one lamella to the other (Edelhauser, et al., 1982; Tripathi et al., 1984; Maurice, 1984), thereby maintaining corneal clarity.
Proteoglycans play an important role in the architectural integrity of these lamellae plates. In vivo the cornea is kept in a dehydrated form by the active aerobic endothelium pump. In hypoxic conditions the epithelium manufactures lactic acid. The acid diffuses into the stroma (Klyce, 1981), resulting in stromal swelling and loss of corneal clarity.
Cornea stored in media with reduced epithelial integrity lose more proteoglycans then those stored with intact epithelium (Slack, et al., 1992). The presence of intact corneal epithelium is required for maintaining the hydration and sodium levels within corneal strome during storage. Loss of epithelium results in increased sodium levels and hydration which may effect post keratoplasty deturgescense. (Jabulinski, 1998) In 1977 it was recognized that if enucle-ation could not be done within one hour of death, corneal epithelium viability could be ensured by placing ice over the eye (Thoft, et al., 1975).
Corneal oxygen uptake was less at the center of transplanted cornea compared to their control other eye (Vannas, et al., 1987). Epithelial permeability, e.g. damage, has been shown to be greater in these transplanted cornea when compared to the control other eye (Chang, et al., 1994). Others have demonstrated significant diminished corneal sensation at the center of the corneal graft (Tugal-tutkun, et al., 1993; Rao, et al., 1985).
In 1974, McCarey & Kaufman realized the addition of glutathione to tissue culture fluid prolonged corneal storage to 72 hours (Roscoe, et al., 1980). Subsequent additions of chondroitin/dextran/and growth factor improved endothelial viability with little effect on corneal epithelium. Chen in 1994 showed the addition of beta-hydroxy butyrate diminished the lactic acid concentration of rabbit cornea by 85% without changing metabolic activity for at least 11 days (Chen, et al., 1994). Subsequent rabbit transplantation after 11 days storage in the Chen media showed corneas to be thin and clear with good endothelium (Chen, et al., 1997).
Beta hydroxybutyrate is an alternative energy source for the krebs cycle. The majority of the metabolism of the corneal metabolism is by the pentose shunt (Bron and Fielder) which in aerobic conditions produces NADPH, ribose and carbon dioxide. Chen has yet to address corneal epithelium issues especially those arising from hypoxia.
An appropriate medium that ensures survival of the corneal cholinergic system of the epithelia (Bron, et al., 1997 and Bron and Fielder) would reduce the likelihood of corneal anesthesia and preserve the viability of the corneal nerves. Preliminary data from perfluorocarbon addition prevents tissue hypoxia. It explains the observation of prolonged epithelial integrity. It may possibly prevent graft anesthesia by keeping the epithelial cholinergic system alive longer. This combination may enable corneal transplantation in other parts of the globe where steroids are not available with minimum medical follow up.
American Eye Banks are recognized as the most efficient in the world (Casey, 1984). Within the United States, approximately 39,000 people are awaiting for an organ transplant (70% for Kidneys). Social pressure has mandated reform for harvesting of tissue. In Pennsylvania, it resulted in act 102. This act made it mandatory that on every death or impending death, a member of the hospital staff had to call Delaware Valley transplant authority (DVTP). The DVTP would then procure organs for harvesting. This came into effect in March 1995. Within six months, Pennsylvania transformed itself from being an importer of cornea to an exporter. This is occurring throughout the nation.
In Europe eye banks developed later and differently. They tissue culture cornea at 33° C. for up to 28 days in MEM and then transport it in Eagles Medium for a further Review of financial data of University Louisville Eye Bank USA and Keratec Eye Bank UK). Redbrake et al. have shown anaerobic conditions to be the rate limiting step in prolonging corneal storage. It would therefore be useful to have an inexpensive storage media that can extend tissue preservation.
The demand for corneal tissue allows private Eye Banks (Keratec) to thrive. The ability to preserve corneal tissue for 35 days would allow the possibility of treating patients worldwide.
Currently no adequate media is available in the United States that can maintain endothelial and epithelial viability for 14 days. Should this become available, it would make the American Eye Banks highly competitive in the increasing global economy.